Perozzo Remo, Folkers Gerd, Scapozza Leonardo
Department of Chemistry and Applied BioSciences, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
J Recept Signal Transduct Res. 2004 Feb;24(1-2):1-52. doi: 10.1081/rrs-120037896.
The understanding of molecular recognition processes of small ligands and biological macromolecules requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures involved. A quantitative description of the forces that govern molecular associations requires determination of changes of all thermodynamic parameters, including free energy of binding (deltaG), enthalpy (deltaH), and entropy (deltaS) of binding and the heat capacity change (deltaCp). A close insight into the binding process is of significant and practical interest, since it provides the fundamental know-how for development of structure-based molecular design-strategies. The only direct method to measure the heat change during complex formation at constant temperature is provided by isothermal titration calorimetry (ITC). With this method one binding partner is titrated into a solution containing the interaction partner, thereby generating or absorbing heat. This heat is the direct observable that can be quantified by the calorimeter. The use of ITC has been limited due to the lack of sensitivity, but recent developments in instrument design permit to measure heat effects generated by nanomol (typically 10-100) amounts of reactants. ITC has emerged as the primary tool for characterizing interactions in terms of thermodynamic parameters. Because heat changes occur in almost all chemical and biochemical processes, ITC can be used for numerous applications, e.g., binding studies of antibody-antigen, protein-peptide, protein-protein, enzyme-inhibitor or enzyme-substrate, carbohydrate-protein, DNA-protein (and many more) interactions as well as enzyme kinetics. Under appropriate conditions data analysis from a single experiment yields deltaH, K(B), the stoichiometry (n), deltaG and deltaS of binding. Moreover, ITC experiments performed at different temperatures yield the heat capacity change (deltaCp). The informational content of thermodynamic data is large, and it has been shown that it plays an important role in the elucidation of binding mechanisms and, through the link to structural data, also in rational drug design. In this review we will present a comprehensive overview to ITC by giving some historical background to calorimetry, outline some critical experimental and data analysis aspects, discuss the latest developments, and give three recent examples of studies published with respect to macromolecule-ligand interactions that have utilized ITC technology.
要理解小分子配体与生物大分子的分子识别过程，需要全面表征结合能，并将热力学数据与相关的相互作用结构进行关联。对控制分子缔合的作用力进行定量描述，需要测定所有热力学参数的变化，包括结合自由能（ΔG）、焓（ΔH）、熵（ΔS）以及热容变化（ΔCp）。深入了解结合过程具有重要的实际意义，因为它为基于结构的分子设计策略的开发提供了基本技术。等温滴定量热法（ITC）是在恒温下测量复合物形成过程中热变化的唯一直接方法。使用这种方法时，将一种结合伙伴滴定到含有相互作用伙伴的溶液中，从而产生或吸收热量。这种热量是可直接观察到的，可通过量热计进行量化。由于灵敏度不足，ITC的应用曾受到限制，但仪器设计的最新进展使得能够测量由纳摩尔（通常为10 - 100）量的反应物产生的热效应。ITC已成为根据热力学参数表征相互作用的主要工具。由于几乎所有化学和生物化学过程中都会发生热变化，ITC可用于众多应用，例如抗体 - 抗原、蛋白质 - 肽、蛋白质 - 蛋白质、酶 - 抑制剂或酶 - 底物、碳水化合物 - 蛋白质、DNA - 蛋白质（等等）相互作用的结合研究以及酶动力学研究。在适当条件下，单次实验的数据分析可得出结合的ΔH、K(B)、化学计量数（n）、ΔG和ΔS。此外，在不同温度下进行的ITC实验可得出热容变化（ΔCp）。热力学数据的信息量很大，并且已经表明它在阐明结合机制方面起着重要作用，并且通过与结构数据的联系，在合理药物设计中也起着重要作用。在本综述中，我们将通过介绍量热法的一些历史背景，对ITC进行全面概述，概述一些关键的实验和数据分析方面，讨论最新进展，并给出三个最近利用ITC技术发表的关于大分子 - 配体相互作用研究的例子。
